The overall goal of this proposal is to understand the mechanism by which Raf Kinase Inhibitory Protein (RKIP) utilizes phosphorylation to switch between three functional states and regulates the cellular kinome. RKIP is the prototypical member of the Phosphatidylethanolamine Binding Protein (PEBP) family, which comprises more than 400 proteins that are evolutionarily conserved both in sequence and structure, spanning from bacteria to humans. In spite of its relatively small size (185 residues), RKIP plays a dual role as a suppressor of metastatic cancer and as a regulator of cardiac function. Importantly, its dysregulation can lead to disease states. In the kinase signaling cascades, RKIP functions both as sensor and effector. As an effector, RKIP modulates allosterically the activity of different kinases, depending on its phosphorylation state. Specifically, RKIP regulates key mammalian signaling cascades, including MAP kinase (MAPK) and G protein- coupled receptors (GPCRs). Phosphorylation by Protein Kinase C (PKC) switches RKIP function from inhibiting Raf/MAPK signaling to inhibiting G-protein-coupled receptor kinase (GRK2), thereby up-regulating the -adrenergic receptor (-AR) and its downstream target Protein Kinase A (PKA). While solid biological data are available, little is known about the molecular mechanisms. We now propose that RKIP functions via a novel regulatory mechanism, where phosphorylation of RKIP acts on an existing salt bridge and triggers an allosteric switch of function. As a sensor, RKIP responds to changes in the MAPK and PKA signal transduction pathways through an unknown mechanism. It has been hypothesized that RKIP would function as a simple two-state system, with RKIP binding to either Raf (RKIPRaf) or GRK2 (RKIPGRK2). However, compelling data from our lab indicate that RKIP adopts three discrete functional and conformational states. The additional, high energy intermediate state (RKIPKin) is the one responsible for the interaction with the kinase cascades. Based on our data, we propose a novel positive feedback loop, where kinases that are downstream of RKIP targets bind and phosphorylate the RKIPKin state. Phosphorylated RKIPKin (pRKIPKin) promotes further phosphorylation of RKIP triggering the phospho-switch. In this proposal, we will characterize the structures and functions of RKIP in its allosteric states, combining our expertise in biophysics and signal transduction. We will accomplish the following specific Aims: 1) Characterize the phospho-switch and the RKIPGRK2 state; 2) Characterize the nature and function of the allosteric switch to a high energy state; and 3) Test the effects of allosteric states defined by biophysical studies on RKIP function in cancer. Although the immediate goal is to understand the signaling role of RKIP, the concepts developed in this grant application will help understand a novel relationship between phosphorylation and allostery that will be of general importance in cell signaling and communication.